The present invention relates to an immunoregulatory molecule that regulates interactions between the molecules involved in costimulatory signal transduction following the first signal transduction involved in the activation of T cells. Said immunoregulatory molecule enables regulation of the immune system. More particularly, the present invention relates to an immunoregulatory molecule having a peptide sequence mimicking a three-dimensional structure of a target molecule wherein said immunoregulatory molecule may be obtained from a phage random peptide library by using a monoclonal antibody which inhibits binding between the molecules involved in costimulatory signal transduction and recognizes a steric structure of said molecules, and a process for preparing said immunoregulatory molecule.
Immunization reaction initiates with the activation of T cells. Antigens, after entering into the living body, are taken up by antigen presenting cells (hereinafter also referred to as xe2x80x9cAPCxe2x80x9d) such as macrophages or B cells. The antigen presenting cells with antigen fragments being incorporated therein then present a complex of antigens (Ag) and major tissue compatibility antigen (MHC) class I or class II on the cellular surface. This MHC/Ag complex is recognized by the T cell receptor (TCR)/CD3 complex on T cells to thereby transmit the first signal into the T cell.
The second signal is also called costimulatory signal and is transmitted into T cells through interaction between the cell adhesion molecules expressed on the cellular membranes of both T cells and APC. Both the first and second signals are necessary for the activation of T cells. It has been observed that inhibition of the second (costimulatory) signal transduction inactivates T cells thereby to lead anergy, i.e. lack of immunological responsiveness. It is believed that the costimulatory signal is transmitted through interaction between CD80 (also called xe2x80x9cB7-1xe2x80x9d or xe2x80x9cB7/BB1xe2x80x9d) and CD86 (also called xe2x80x9cB7-2xe2x80x9d or xe2x80x9cB70xe2x80x9d) expressed on APC and their corresponding receptors expressed on T cells, CD28 and cytotoxic T lymphocyte associated antigen (CTLA-4) (Lenschow, D. J. et al., Annu. Rev. Immunol. 14, 233-258, 1996).
CD28 molecule is a glycoprotein expressed on T cells. It is elucidated that CD28 molecule acts as a costimulatory receptor which promotes T cell growth and production of various cytokines upon stimulation from TCR. CTLA-4 molecule is known to be quite similar to CD28 in both its structure and function. In CTLA-4-knockout mice, T cells proliferate in the spleen or lymph nodes, T cell infiltration occurs in many organs including cardiac muscle, thyroid, spleen, etc. and the mice die of autoimmune diseases, suggesting that CTLA-4 is a negative regulator of T cell response.
As a ligand of CD28/CTLA-4, Linsley et al. identified CD80 molecule in 1990 (Proc. Natl. Acad. Sci USA, 87, 5031-5035) and Azuma et al. identified CD86 molecule in 1993 (Nature, 366, 76-79). CD80 is a transmembrane glycoprotein with a short intracellular region having a signal transduction domain which is classified into Ig superfamily. Like CD80, CD86 is also a transmembrane glycoprotein classified into Ig superfamily. However, unlike CD80, CD86 bears three domains of potential protein kinase C dependent phosphorylation in the intracellular region, suggesting that it is capable of signal transduction. Both CD80 and CD86 are involved in growth, cytotoxic activity and differentiation in the thymus, of T cells. CD28 or CTLA-4 molecule has an ability to bind to CD80 or CD86 molecule and it is believed that the stages when these molecules are expressed or interaction of these molecules may induce the activation or inhibition of T cells to ultimately affect a whole immune system.
As described above, CD28, CTLA-4, CD80 and CD86 molecules play an extremely important role in exerting various immunological functions such as antigen response of T cells or interactions of B cells. These molecules undertake major parts of the costimulatory signal transduction following the first signal transduction through interaction between MHC/Ag complex and TCR/CD3 complex. Thus, one can expect that a whole immune system may be regulated through regulation of the costimulatory signal transduction. As one of such attempts, antibodies to CD28 and CTLA-4 molecules were used wherein T cells previously stimulated with anti-CD3 antibody were reacted with anti-CTLA-4 or anti-B7 antibodies in the presence of anti-CD28 antibody thereby to enhance the activation of T cells by anti-C28 antibody by a little less than 3-fold (Matthew F. et al., J. Exp. Med. 182, 459-465, 1995).
Transduction of information within the living body is made as a signal transduction mediated by binding between a receptor molecule expressed on the cellular surface and its corresponding ligand molecule. Receptor and ligand molecules are complementary to each other and can bind together when they are in a key-and-keyhole relationship. However, the most crucial site involved in binding resides within a small portion of ligand. Thus, elucidation of the structure of said site for interaction enables inhibition or enhancement of binding between receptor and ligand.
To elucidate the structure of either of sites for interaction in a certain receptor-ligand system, a peptide scanning procedure has conventionally been used wherein peptides of an appropriate size are synthesized over a whole length of amino acid sequence of said protein and are studied for their binding capacity or binding inhibitory activity to screen the site for interaction. Alternatively, contribution of each amino acid residue to interaction was determined by one-by-one amino acid substitution by site-directed mutagenesis, or amino acid residues involved in protein-protein interaction were identified by photoaffinity procedure.
More recently, there has been used a screening with a phage random peptide library as developed by Scott et al. (Science, 249, 386-390, 1990) wherein genes encoding random peptide molecules are inserted into the gene (geneIII) of pIII molecule being consisted of phage, pIII molecule are expressed together with random peptide molecules incorporated therein on fd-tet phage (filamentous single stranded DNA phage), a phage random peptide library is prepared, phages bound are selected by, for example, panning, and said pIII gene is sequenced to determine the interaction site. According to this procedure, a population of as many as several 105 kinds of peptides may be obtained more easily than chemical synthesis and moreover amplification is also possible as required.
Using a phage random peptide library, there has been attempted to identify interaction sites on a target molecule. Typically, a ligand or a receptor is directly bound to a plate as a solid phase and a phage random peptide library is screened for desired interaction sites by panning. However, this procedure provides a large number of phage clones capable of binding to the target molecule, most of which phage clones however cannot bind to the critical binding site that regulates a ligand-receptor interaction. Thus, those phages having a peptide sequence capable of regulating a ligand-receptor interaction could only be obtained with much difficulty and poor efficiency.
When a phage library is screened with antibodies having definite antigen specificity, effective binding peptides have been obtained comparatively efficiently. For example, for determining epitopes on p53 molecule, anti-p53 monoclonal antibody was used with a random peptide library of various lengths of amino acid sequence wherein peptides having an amino acid sequence highly homologous to that of p53 molecule are screened with anti-p53 monoclonal antibody and therefrom the epitopes were identified (Stepheni, C. W. et al., J. Mol. Biol., 248, 58-78, 1995). Also, a phage random peptide library of a sequence of six amino acid residues was screened with anti-bFGF antibody to give peptides having similar amino acid sequence to that of bFGF. It was reported that synthetic peptides prepared by elongating these screened peptides to those having eight amino acid residues inhibited binding between bFGF and its receptor FGFR-1 (Yayon, A. et al., Proc. Natl. Acad. Sci. USA, 90, 10643-10647, 1993). When motifs capable of binding to antibodies recognizing conformational epitopes on acetylcholine receptor were screened with a phage random peptide library of a sequence of six amino acid residues, peptides were obtained which had a non-homologous amino acid sequence to that of acetylcholine receptor but were capable of binding to this antibody. However, whether these peptides bind to acetylcholine is not referred to (Balass, M. et al., Proc. Natl. Acad. Sci. USA, 90, 10638-10642, 1993).
As mentioned hereinabove, in order to regulate the immune reaction through regulation of molecules involved in costimulatory signal transduction, antibodies to B7 or CTLA-4 molecules have been used. However, the effect of these antibodies, i.e. enhancement in the activation of T cells by a little less than 3-fold, is not sufficiently high. Furthermore, no specific antigens were used in their experiment and hence the effect was not proved in in vivo immune reaction. Other than antibodies, there is no report to attempt to design small molecules involved in costimulatory signal transduction for use as a medicament.
For designing such small molecules, the conventional approach was to first analyze sites associated with interaction of target molecules and to synthesize peptides based on the determined amino acid sequence of said sites to thereby design desired small molecules. However, even if an amino acid sequence of sites associated with protein-protein interaction is determined by the conventional analytical procedure and oligopeptides are prepared based on the obtained primary amino acid sequence, it was difficult to design small molecules which can act in replace of target molecules. That is, sites associated with interaction of protein molecules having crucial function are defined by a three-dimensional structure and hence amino acid residues involved in interaction are dispersed within a primary amino acid sequence although they gather together as a three-dimensional structure is constructed. Thus, from oligopeptides prepared based on a primary amino acid sequence of sites associated with interaction, reconstruction of a three-dimensional structure of said sites was technically restricted and designing small molecules with effective function was difficult.
In case of screening with a phage random peptide library, screening by panning with a receptor or a ligand directly bound to a solid phase could provide those phages having a specific peptide sequence capable of regulating receptor-ligand interaction only with quite poor efficiency and difficulty. The screening with a protein molecule having a relatively definite antigen specificity such as an antibody is merely used for determining a primary amino acid sequence of a target molecule to which said antibody binds or for determining motifs having high homology with said primary amino acid sequence. In the absence of such homology, however, peptide sequences to which the antibody could merely bind were obtained. That is, there has been no report that molecules mimicking a three-dimensional structure of a target molecule to which an antibody binds could be obtained without depending on a primary amino acid sequence thereof. Much less, there is no report that small molecules capable of regulating the immune system could be obtained by targeting CD80 and CD86 expressed on APC and their corresponding receptors, CD28 and CTLA-4 expressed on T cells. Also, there is no report that such small molecules could be designed without depending on a primary amino acid sequence of the target molecules.
The present invention provides an immunoregulatory molecule that regulates interactions between molecules involved in costimulatory signal transduction following the first signal transduction mediated by MHC/Ag complex and TCR/CD3 complex involved in the activation of T cells. Said immunoregulatory molecule enables regulation of the immune system. More particularly, the present invention provides an immunoregulatory molecule having a peptide sequence mimicking a three-dimensional structure of a target molecule wherein said immunoregulatory molecule inhibits binding between molecules involved in costimulatory signal transduction and may be obtained from a phage random peptide library with the use of a monoclonal antibody recognizing a steric structure of said molecules, and a process for preparing said immunomodulating molecule.